The high mass sensitivity identification and quantitation of large numbers of peptides from protein digests is a major goal in proteomics. Nanoflow liquid chromatography, using commercially available 75 and 100 μm i.d. reversed phase columns, offers the advantages of high resolution, high mass sensitivity and low sample and mobile phase consumption. However, analysis of a limited amount of sample (e.g., tumor biopsy, laser capture microdissected cells, immunoprecipitated proteins, 2-D gel spots, etc.) can still be challenging with the above columns. For a fixed limited amount of sample injected, columns with smaller inner diameter can decrease chromatographic band dilution (Novotny et al., 1985; Haskins et al., 2001; and Unger, 1990) and thus increase the signal for concentration-sensitive ESI-MS (Shen et al., 2000). However, narrow bore columns (particularly those having less than 50 μm i.d.) are difficult to pack with conventional microparticles of 1-5 μm because of the very high pressure required to overcome the low column permeability due to the narrow bore (Shen et al., 2000; MacNair et al., 1999; and Wu et al., 2001).
A monolithic separation medium is made of a continuous, rigid, porous polymeric rod. Typically, prior art polymeric monolithic capillary columns for nano-LC and CEC separations, such as are disclosed in U.S. Patent Application No. US 2002/0088753 (Huber et al., 2002), which is hereby incorporated by reference herein, have been made in capillaries having inner diameters larger than 100 μm (Premstaller et al., 2001; Premstaller et al., 2000; Svec et al., 1995; Tennikov et al., 1998; Petro et al., 1996; Moore et al., 1998; Adam et al., 2000 (a); Adam et al., 2000 (b); and Enlund et al., 2001). Several approaches for synthesis of polymeric monoliths in capillaries of 100-300 μm i.d. have been published in which the reaction mixture is placed in the capillary, followed by UV- or thermally-induced in situ polymerization (Premstaller et al., 2001; Huang et al., 2002; Xie er al., 1999, Moore et al., 1998; Petro et al., 1996; Myers et al., 1999; and Rohr et al., 2003). High efficiencies are found, using the narrow bore capillary columns that have been made, due to decreased flow dispersion and a homogeneous packing bed structure. However, it would be very desirable to be able to extend those advantages consistently to significantly more narrow i.d. columns, particularly to those having i.d. values of 25 μm or less.